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Sample GSM3904479 Query DataSets for GSM3904479
Status Public on Jun 28, 2019
Title DamID, Dam, F121-9, TALE-VP64, Nrp1, rep1
Sample type SRA
 
Source name DamID, Dam, F121-9, TALE-VP64, Nrp1
Organism Mus musculus
Characteristics cell line: F121-9
genotype/cell type: F121-9 mESC
Treatment protocol RPE-1 cells: sgRNAs were cloned into Lentiguide-Puro vector (Addgene #52963) using restriction enzyme BsmBI, and Lentivirus was prepared. RPE-1 cells stably expressing SunTag-CRISPRa were infected with LentiGuide virus and selected with 10 µg/µl Puromycin for one week to obtain stable polyclonal cell pools.
Growth protocol Cells were cultured according to 4DNucleome SOP, https://www.4dnucleome.org/cell-lines.html
Extracted molecule genomic DNA
Extraction protocol DamID-seq was performed as described in (Brueckner et al. 2016) with slight modifications. Instead of plasmid transfections, Dam-LMNB1 or Dam were added by Lentiviral transduction. Three days after infection, cells were collected for gDNA isolation. gDNA was pre-treated with SAP (10 U, New England Biolabs) in CutSmart buffer in a total volume of 10 µl at 37 °C for 1 h followed by heat inactivation at 65°C for 20 min to remove signal from apoptotic fragments. gDNA was digested with DpnI (10 U, New England Biolabs) in CutSmart buffer in a total volume of 10 µl at 37 °C for 8 h followed by heat inactivation at 80 °C for 20 min. Fragments were ligated to 12.5 pmol DamID adapters using T4 ligase (2.5 U, New England Biolabs) in T4 ligase buffer in a total volume of 20 µl incubated at 16 °C for 16 h. The reaction was heat inactivated for 10 min at 65 °C. Products were then digested with DpnII to remove partially methylated fragments. DpnII buffer and DpnII (10 U, New England Biolabs) were added in a total volume of 50 µl and incubated at 37 °C for 1 h. 8 µl of DpnII-digested products was amplified by PCR with MyTaq Red Mix (Bioline) and 2.5 µM primers Adr-PCR-Rand1 in a total volume of 40 µl. PCR settings were 8 min at 72 °C (1×) followed by 20 s at 94 °C, 30 s at 58 °C, 20 s at 72 °C (24× for Dam, 28x for DamLMNB1 samples) and 2 min at 72 °C (1×). Remaining steps were performed as previously described.
DamID-seq was performed as described in (Brueckner et al. 2016) with slight modifications. Instead of plasmid transfections, Dam-LMNB1 or Dam were added by Lentiviral transduction. Three days after infection, cells were collected for gDNA isolation. gDNA was pre-treated with SAP (10 U, New England Biolabs) in CutSmart buffer in a total volume of 10 µl at 37 °C for 1 h followed by heat inactivation at 65°C for 20 min to remove signal from apoptotic fragments. gDNA was digested with DpnI (10 U, New England Biolabs) in CutSmart buffer in a total volume of 10 µl at 37 °C for 8 h followed by heat inactivation at 80 °C for 20 min. Fragments were ligated to 12.5 pmol DamID adapters using T4 ligase (2.5 U, New England Biolabs) in T4 ligase buffer in a total volume of 20 µl incubated at 16 °C for 16 h. The reaction was heat inactivated for 10 min at 65 °C. Products were then digested with DpnII to remove partially methylated fragments. DpnII buffer and DpnII (10 U, New England Biolabs) were added in a total volume of 50 µl and incubated at 37 °C for 1 h. 8 µl of DpnII-digested products was amplified by PCR with MyTaq Red Mix (Bioline) and 2.5 µM primers Adr-PCR-Rand1 in a total volume of 40 µl. PCR settings were 8 min at 72 °C (1×) followed by 20 s at 94 °C, 30 s at 58 °C, 20 s at 72 °C (24× for Dam, 28x for DamLMNB1 samples) and 2 min at 72 °C (1×). Remaining steps were performed as previously described.
 
Library strategy OTHER
Library source genomic
Library selection other
Instrument model Illumina HiSeq 2500
 
Description 4591_1_F121-9_Nrp1_Dam_r1_ACAAGCTA_S2
Data processing Data analysis of DamID samples: First, the constant DamID adapter was trimmed from the 65 bp single-end reads (with cutadapt 1.11 and custom scripts). The remaining gDNA starting with GATC was mapped to hg19 with bowtie2 2.2.6. Uniquely mapped reads (filtered for bowtie’s XS-tag) were counted to GATC fragments.
Genome_build: hg19 for homo sapiens samples, mm10 for mus musculus samples
 
Submission date Jun 25, 2019
Last update date Jun 29, 2019
Contact name Bas van Steensel
E-mail(s) b.v.steensel@nki.nl
Phone + 31 20 512 2040
Fax +31 20 669 1383
URL http://www.nki.nl/nkidep/vansteensel
Organization name Netherlands Cancer Institute
Department division of Molecular Biology
Lab van Steensel group
Street address Plesmanlaan 121
City Amsterdam
ZIP/Postal code 1066 CX
Country Netherlands
 
Platform ID GPL17021
Series (2)
GSE133272 Effects of transcription on genome - nuclear lamina interactions: DamID-seq data
GSE133275 Effects of transcription on genome - nuclear lamina interactions
Relations
BioSample SAMN12136105
SRA SRX6365979

Supplementary file Size Download File type/resource
GSM3904479_F121-9_Nrp1_Dam_r1-gatc.counts.txt.gz 42.3 Mb (ftp)(http) TXT
SRA Run SelectorHelp
Raw data are available in SRA
Processed data provided as supplementary file

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